概述

Fluo-8 No Wash Calcium Assay Kit (ab112129) is a fluorescence-based assay for detecting intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fluo-8 which can cross the cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-8 are cleaved by an esterase, resulting in a negatively charged fluorescent dye that stays inside the cell. Its fluorescence is greatly enhanced upon binding to calcium. When cells are stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increases the fluorescence of Fluo-8. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement make Fluo-8 the brightest green calcium indicator available in the marker, and it is an ideal tool for the measurement of cellular calcium through HTS screening.

ab112129 provides an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels using HTS instrumentation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

This product is intended to be used for monitoring calcium fluctuations in vivo in live cells using the following HTS imaging plate readers: FLIPR™, FDSS, BMG NOVOstar™, FLexStation, ViewLux, IN Cell Analyzer or Arrayscan.

Readers with an in built pipetter are essential for measuring calcium response accurately due to transient property of the calcium signal. Depending on dose of the agonist, the peak response is few seconds to 20 seconds, so one will need to add the compound by instrument and read the fluorescent signal simultaneously every second for ~1-2 min.

相关性Calcium is essential for all living organisms, where Ca2+ sequestration and release into and out of the cytoplasm functions as a signal for many cellular processes. 99% of calcium is found in bones and teeth with the remaining 1% found in the blood and soft tissue. Serum calcium levels are tightly controlled (8.4-11.4 mg/dL) and any variation outside this range can have serious effects. Calcium plays a role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. Calcium ion channels control the migration of calcium ions across cell membranes, permitting the activation and inhibition of a wide variety of enzymes. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels that can occur in severe alcoholism.

应用

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用

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说明

Functional Studies

Use at an assay dependent concentration.

Fluo-8 No Wash Calcium Assay kit 图像

Functional Studies - Fluo-8 No Wash Calcium Assay kit (ab112129)

Carbachol Dose Response was measured in HEK293 cells with ab112129 and a Fluo-4 calcium assay kit. HEK293 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using the ab112129 or the Fluo-4 kit for 1 hour at room temperature. Carbachol (50 µL/well) was added to achieve the final concentrations indicated.

The Ca response is a transient response (a couple of the seconds to 10-20 seconds), it is almost impossible to pipette the compounds into the plate, and read the response. Yes, you do use the multichannel pipette to add the dye into the plate, and let ...

I realize that there was a typos in my last email. The kD value I should have written is 320nm. However I also contacted laboratory after receiving your reply - they have informed me that the kD value is 389...

It seems that you have asked similar question last year in October and we provided the 230nm as dissociation constant. The 230 nm Kd value is available to us from our collaborator who developed this product for us....